Deubiquitylating Enzyme USP1 As Therapeutic Target in Multiple Myeloma

Introduction Deregulation of the ubiquitin-proteasome system (UPS) is linked to pathogenesis of various human diseases, including cancer. Targeting the proteasome is an effective therapy in multiple myeloma (MM) patients.Recent research efforts led to the discovery of newer agents that target enzymes modulating protein ubiquitin- conjugation/deconjugation rather than the proteasome itself, with the goal of generating more specific and less toxic anti-tumor therapies. Our prior studies have identified a role of deubiquitylating enzymes (DUBs) USP7, USP14, and UCHL5 in MM pathogenesis, and provided the rationale for targeting these DUBs in MM (Chauhan et al., Cancer Cell 2012, 11:345-358). Among DUBs, USP1 regulates DNA repair and the Fanconi anemia pathway by deubiquitylating two critical DNA repair proteins, FANCD2-Ub and PCNA-Ub. Additionally, USP1 stabilizes tumor-promoting inhibitor of DNA binding (ID) proteins. Here we examined the role of USP1 DUB in MM using both biochemical and RNA interference strategies.

Methods We utilized MM cell lines, patient cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors. Cell viability was assessed using WST and CellTiter-Glo assays. MM.1S cells were transiently transfected with control short interfering RNA (siRNA) USP1 ON TARGET plus SMART pool siRNA using the cell line Nucleofector Kit V. A biochemical inhibitor of USP1 SJB3-019A (SJB) was purchased from Medchem Express, USA. In vitro DUB enzymatic activity was assessed using Ubiquitin-AMC and Ubiquitin-Rhodamine assay kits, as well as Ub-CHOP-reporter and K48-linked Ubiquitin tetramers. Competitive Ub-VS probe labeling was performed, as previously described (Chauhan et al., Cancer Cell 2012, 11:345-358). Signal transduction pathways were evaluated using immunoblotting. Statistical significance of data was determined using a Student's t test.

Results Gene expression profiling (GEP) analysis of USP1 showed significantly higher USP1 levels in patient MM cells versus normal plasma cells (p < 0.05).We found a statistically significant inverse correlation between USP1 levels and overall patient survival (p =0.036). Immunoblot blot analysis show higher USP1 levels in MM cell lines and patient cells compared to normal cells.USP1 knock-down reduced MM cell viability (p < 0.05).To validate our siRNA data, we utilized a novel USP1 inhibitor SJB3-019A (SJB). Analysis using Ub-Rhodamine, Ub-AMC and Ub-E_KL reporter assays showed that SJB is a potent, specific, and selective inhibitor of cellular USP1 DUB activity (EC50=1μM), and does not inhibit other DUBs (USP2/USP5/USP7/USP14) or other families of cysteine proteases (UCH37) (EC50 >10 μM). SJB blocks labeling of USP1 with HA-Ub-VS probe in a concentration-dependent manner, but did not alter labeling of other DUBs with HA-Ub-VS. SJB inhibits USP1-mediated cleavage of K48-linked Tetra-ubiquitin chains, but not that mediated by USP2 or USP7. Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, Dox-40, ARP1, KMS11, U266, ANBL6.WT, ANBL6.BR and LR5) and primary patient cells for 24h significantly decreases their viability (IC₅₀ range 100nM to 500nM) (p< 0.05; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting selective anti-MM activity and a favorable therapeutic index for SJB. Tumor cells from 4 patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies were sensitive to SJB. Furthermore, SJB3-019A inhibits proliferation of MM cells, even in the presence of BM stromal cells and plasmacytoid dendritic cells. Mechanistic studies show that SJB3-019A- triggered apoptosis is associated with 1) induction of cell cycle arrest via p21 upregulation; 2) activation of caspase 3, caspase-8 and caspase-9; 3) decreased homologous recombination activity and increased levels of Ub-FANCD2, Ub-FANCI, and Ub-PCNA; 4) defective DNA repair, evident by reduced RAD51; 5) degradation of USP1 and ID proteins; and 6) downregulation of Notch-1, Notch-2, SOX-4, and SOX-2 proteins. Finally, combination of SJB with lenalidomide, pomalidomide, HDACi ACY-241, or bortezomib induces synergistic anti-MM activity and overcomes drug resistance.

Conclusion Our preclinical studies provide the framework for clinical evaluation of USP1 inhibitors, alone or in combination, as a potential novel MM therapy.